Means for the modulation of processes mediated by retinoid receptors and compounds useful therefor

ABSTRACT

In accordance with the present invention, there are provided methods to modulate processes mediated by retinoid receptors, employing high affinity, high specificity ligands for such receptors. In one aspect of the present invention, there are provided ligands which are more selective for the retinoid X receptor than is retinoic acid (i.e., rexoids). In another aspect of the present invention, alternative ligands (other than retinoic acid) have been discovered which are capable of inducing retinoic acid receptor mediated processes. In yet another aspect, methods have been developed for the preparation of such retinoid receptor ligands from readily available compounds.

RELATED APPLICATIONS

This application is a divisional of Ser. No. 09/350,648, filed Jul. 9,1999, now U.S. Pat. No. 6,576,676, which is a divisional of Ser. No.08/472,817, filed Jun. 7, 1995, now U.S. Pat. No. 5,968,989, which is adivisional of Ser. No. 08/244,857, filed on Jun. 14, 1994, now pendingwhich is a 371 and based on PCT/US92/11214, filed Dec. 18, 1992, whichis a continuation-in-part of Ser. No. 07/908,980, filed Dec. 18, 1991,now abandoned.

FIELD OF THE INVENTION

The present invention relates to intracellular receptors, and ligandstherefor. In a particular aspect, the present invention, relates tomethods for modulating processes mediated by retinoid receptors.

BACKGROUND OF THE INVENTION

A central problem in eukaryotic molecular biology continues to be theelucidation of molecules and mechanisms that mediate specific generegulation in response to exogenous inducers such as hormones or growthfactors. As part of the scientific attack on this problem, a great dealof work has been done in efforts to identify exogenous inducers whichare capable of mediating specific gene regulation.

Although much remains to be learned about the specifics of generegulation, it is known that exogenous inducers modulate genetranscription by acting in concert with intracellular components,including intracellular receptors and discrete DNA sequences known ashormone response elements (HREs).

As additional members of the steroid/thyroid superfamily of receptorsare identified, the search for exogenous inducers for such newlydiscovered receptors (i.e., naturally occurring (or synthetic) inducers)has become an important part of the effort to learn about the specificsof gene regulation.

The retinoid members of the steroid/thyroid superfamily of receptors,for example, are responsive to compounds referred to as retinoids, whichinclude retinoic acid, retinol (vitamin A), and a series of natural andsynthetic derivatives which have been found to exert profound effects ondevelopment and differentiation in a wide variety of systems.

The identification of compounds which interact with retinoid receptors,and thereby affect transcription of genes which are responsive toretinoic acid (or other metabolites of vitamin A), would be ofsignificant value, e.g., for therapeutic applications.

Recently, a retinoic acid dependent transcription factor, referred to asRAR-alpha (retinoic acid receptor-alpha), has been identified.Subsequently, two additional RAR-related genes have been isolated; thusthere are now at least three different RAR subtypes (alpha, beta andgamma) known to exist in mice and humans. These retinoic acid receptors(RARs) share homology with the superfamily of steroid hormone andthyroid hormone receptors and have been shown to regulate specific geneexpression by a similar ligand-dependent mechanism [Umesono et al.,Nature 336: 262 (1988)]. These RAR subtypes are expressed in distinctpatterns throughout development and in the mature organism.

More recently, additional novel members of the steroid/thyroidsuperfamily of receptors have been identified, such as, for example,retinoid X receptor-alpha [RXR-α; see Mangelsdorf et al., in Nature 345:224–229 (1990)], retinoid X receptor-beta [RXR-β; see Hamada et al.,Proc. Natl. Acad. Sci. USA 86: 8289–8293 (1989)], and retinoid Xreceptor-gamma [RXR-γ; see Mangelsdorf et al., Genes and Development6:329–344 (1992)]. While these novel receptors are responsive toretinoic acid, the primary exogenous inducer(s) for these receptors havenot been identified.

Although both RAR and RXR respond to retinoic acid in vivo, thereceptors differ in several important aspects. First, RAR and RXR aresignificantly divergent in primary structure (e.g., the ligand bindingdomains of RARα and RXRα have only 27% amino acid identity). Thesestructural differences are reflected in different relative degrees ofresponsiveness of RAR and RXR to various vitamin A metabolites andsynthetic retinoids. In addition, distinctly different patterns oftissue distribution are seen for RAR and RXR. In contrast to the RARs,which are not expressed at high levels in the visceral tissues, RXRαmRNA has been shown to be most abundant in the liver, kidney, lung,muscle and intestine. Finally, response elements have recently beenidentified in the cellular retinol binding protein type II (CRBPII) andapolipoprotein AI genes which confer responsiveness to RXR, but not RAR.Indeed, RAR has also been recently shown to repress RXR-mediatedactivation through the CRBPII RXR response element. These data, inconjunction with the observation that both RAR and RXR can activatethrough the RAR response element of the RARβ promoter, indicate that thetwo retinoic acid responsive pathways are not simply redundant, butinstead manifest a complex interplay.

In view of the related, but clearly distinct nature of these receptors,the identification of ligands which are more selective for the retinoidX receptor than is retinoic acid would be of great value in selectivelycontrolling processes mediated by one or both of these retinoid receptortypes.

Other information helpful in the understanding and practice of thepresent invention can be found in commonly assigned, co-pending U.S.patent application Ser. Nos. 108,471, filed Oct. 20, 1987 (now issued asU.S. Pat. No. 5,071,773); 276,536, filed Nov. 30, 1988 (now issued asU.S. Pat. No. 4,981,784); 325,240, filed Mar. 17, 1989; 370,407, filedJun. 22, 1989; and 438,757, filed Nov. 16, 1989, all of which are herebyincorporated herein by reference in their entirety.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, we have developed methods tomodulate retinoid receptor mediated processes, employing high affinity,high specificity ligands for such receptors.

In a particular aspect of the present invention, there are providedligands which are high affinity, high specificity ligands for retinoidreceptors. Thus, in one aspect of the present invention, there areprovided ligands which are more selective for the retinoid X receptorthan is all-trans-retinoic acid. In another aspect of the presentinvention, we have discovered alternative ligands (other thanall-trans-retinoic acid) which are capable of inducing retinoic acidreceptor mediated processes.

In yet another aspect of the present invention, we have developedmethods for the preparation of such retinoid receptor ligands fromreadily available retinoid compounds.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a transactivation profile of various HPLC fractions obtainedfrom retinoic acid (RA)-treated S2 cells.

FIG. 2 a is a comparison of the transactivation profile ofall-trans-retinoic acid (RA) on RAR-alpha and RXR-alpha.

FIG. 2 b is a similar comparison to that shown in FIG. 2 a, employingHPLC fraction 18 (instead of RA).

FIG. 3 presents several activation profiles for analysis of RXR-alpha orRAR-alpha activation by various retinoic acid isomers. Panel a.represents experiments done in insect S2 cells, while panels b. and c.represent experiments done in mammalian CV-1 cells. In the figure,closed circles are used to designate 9-cis-retinoic acid, open circlesare used for all-trans-retinoic acid, open triangles are used for13-cis-retinoic acid and open squares are used for 11-cis-retinoic acid.

FIG. 4 presents the results of saturation binding analysis of9-cis-retinoic acid. Cell extracts were incubated with increasingconcentrations of tritiated retinoid in the absence (total binding) orpresence (non-specific binding) of 200-fold excess non-tritiatedretinoid. Non-specific binding was subtracted from total binding andplotted as specific binding. The data shown in FIG. 4 a representspecific [³H]-9-cis-retinoic acid binding to RXRα (closed circles) ormock (open circles) extracts; or specific [³−H]-all-trans-retinoic acidbinding to RXRα (open squares).

FIG. 4 b presents a Scatchard analysis, wherein specific 9-cis-retinoicacid binding to RXRα in (a) was transformed by Scatchard analysis andplotted. Linear regression yielded a Kd=11.7 nM (r=0.86).

FIG. 5 presents a DNA-cellulose column profile of radiolabelled9-cis-retinoic acid bound to baculovirus expressed RXR. In FIG. 5 a,sample cell extracts containing RXRα protein were labelled with 10 nM[³H]-9-cis-retinoic acid in the absence (open squares) or presence (opencircles) of 200-fold excess non-radioactive 9-cis-retinoic acid, andthen applied to the DNA-cellulose column. Fall-through radioactivity wasmonitored until a consistent baseline was established. DNA-bindingcomponents were then eluted with a linear salt gradient. The peakradioactive fractions (labelled 1–15) were then subjected to immunoblotanalysis using an hRXRα-specific antisera. The peak radioactive fraction(indicated by an arrow) co-migrated exactly with the peak amount ofRXRα-specific protein.

In FIG. 5 b, the peak radioactive fraction of the DNA-cellulose columnis shown to contain 9-cis-retinoic acid. The peak fraction (arrow in(a)) was extracted and analyzed on a C₁₈ column developed with mobilephase G. As shown, 0.95% of the extracted radioactivity co-elutes withauthentic 9-cis-retinoic acid (absorbance peak).

FIG. 6 is a comparison of the transactivation profile for RXR-alpha inthe presence of 9-cis-retinoic acid employing a luciferase reportercontaining the retinoid response element derived from either theapolipoprotein A1 gene (APOA13) or cellular retinol binding protein,type II (CRBPII).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided a method formodulating process(es) mediated by retinoid receptors, said methodcomprising conducting said process(es) in the presence of at least onecompound of the structure:

wherein:

unsaturation between carbon atoms C⁹ and C¹⁰ has a cis configuration,and one or both sites of unsaturation between carbon atoms C¹¹ throughC¹⁴ optionally have a cis configuration;

“Ring” is a cyclic moiety, optionally having one or more substituentsthereon;

Z is selected from carboxyl (—COOH), carboxaldehyde (—COH), hydroxyalkyl[—(CR′₂)_(n)—OH, wherein each R′ is independently selected from hydrogenor a lower alkyl and n falls in the range of 1 up to about 4], thioalkyl[—(CR′₂)_(n)—SH, wherein R′ and n are as defined above], hydroxyalkylphosphate [—(CR′₂)_(n)—OP(OM)₃, wherein R′ and n are as defined aboveand M is hydrogen, lower alkyl, or a cationic species such as Na⁺, Li⁺,K⁺, and the like], alkyl ether of a hydroxyalkyl group [—(CR′₂)_(n)—OR′,wherein R′ and n are as defined above], alkyl thioether of a thioalkylgroup [—(CR′₂)_(n)—SR′, wherein R′ and n are as defined above], estersof hydroxyalkyl groups [—(CR′₂)_(n)—O—CO—R′, wherein R′ and n are asdefined above], thioesters of hydroxyalkyl group [—(CR′₂)_(n)—O—CS—R′,wherein R′ and n are as defined above], esters of thioalkyl groups[—(CR′₂)_(n)—S—CO—R′, wherein R′ and n are as defined above], thioestersof thioalkyl groups [—(CR′₂)_(n)—S—CS—R′, wherein R′ and n are asdefined above], aminoalkyl [—(CR′₂)_(n)—NR′₂, wherein R′ and n are asdefined above], N-acyl aminoalkyl [—(CR′₂)_(n)—NR′—CO—R″, wherein R′ andn are as defined above and R″ is a lower alkyl or benzyl], carbamate[—(CR′₂)_(n)—NR′—CO—OR′ or —(CR′₂)_(n)—O—CO—NR′₂, wherein R′ and n areas defined above], and the like; and

each R is independently selected from H, halogen, alkyl, aryl, hydroxy,thiol, alkoxy, thioalkoxy, amino, or any of the Z substituents, and thelike; or

any two or more of the R groups can be linked to one another to form oneor more ring structures.

Exemplary R groups in the latter situation are selected from alkylene,oxyalkylene, thioalkylene, and the like.

As employed herein, the term “modulate” refers to the ability of aligand for a member of the steroid/thyroid superfamily to induceexpression of gene(s) maintained under hormone expression control, or torepress expression of gene(s) maintained under such control.

As employed herein, the phrase “processes mediated by retinoidreceptors” refers to biological, physiological, endocrinological, andother bodily processes which are mediated by receptor or receptorcombinations which are responsive to natural or synthetic retinoids, ornatural or synthetic compounds as defined herein (referred to herein as“rexoids” because of the ability of many of the compounds describedherein to selectively activate retinoid X receptors). Modulation of suchprocesses can be accomplished in vitro or in vivo. In vivo modulationcan be carried out in a wide range of subjects, such as, for example,humans, rodents, sheep, pigs, cows, and the like.

Exemplary receptors which are responsive to retinoids, and natural orsynthetic compounds as defined herein (i.e., “rexoids”), includeretinoic acid receptor-alpha, retinoic acid receptor-beta, retinoic acidreceptor-gamma, and splicing variants encoded by the genes for suchreceptors; retinoid X receptor-alpha, retinoid X receptor-beta, retinoidX receptor-gamma, and splicing variants encoded by the genes for suchreceptors; as well as various combinations thereof (i.e., homodimers,homotrimers, heterodimers, heterotrimers, and the like), includingcombinations of such receptors with other members of the steroid/thyroidsuperfamily of receptors with which the retinoid receptors may interactby forming heterodimers, heterotrimers, and higher heteromultimers. Forexample, the retinoic acid receptor-alpha may form a heterodimer withretinoid X receptor-alpha, the retinoic acid receptor-beta may form aheterodimer with retinoid X receptor-alpha, retinoic acid receptor-gammamay form a heterodimer with retinoid X receptor-alpha, retinoid Xreceptor-alpha may form a heterodimer with thyroid receptor, retinoid Xreceptor-beta may form a heterodimer with vitamin D receptor, retinoid Xreceptor-gamma may form a heterodimer with retinoic acid receptor-alpha,and the like.

As employed herein, the phrase “members of the steroid/thyroidsuperfamily of receptors” (also known as “nuclear receptors” or“intracellular receptors”) refers to hormone binding proteins thatoperate as ligand-dependent transcription factors, including identifiedmembers of the steroid/thyroid superfamily of receptors for whichspecific ligands have not yet been identified (referred to hereinafteras “orphan receptors”). These hormone binding proteins have theintrinsic ability to bind to specific DNA sequences. Following binding,the transcriptional activity of target gene (i.e., a gene associatedwith the specific DNA sequence), is modulated as a function of theligand bound to the receptor.

The DNA-binding domains of all of these nuclear receptors are related,consisting of 66–68 amino acid residues, and possessing about 20invariant amino acid residues, including nine cysteines.

A member of the superfamily can be identified as a protein whichcontains the above-mentioned invariant amino acid residues, which arepart of the DNA-binding domain of such known steroid receptors as thehuman glucocorticoid receptor (amino acids 421–486), the estrogenreceptor (amino acids 185–250), the mineralocorticoid receptor (aminoacids 603–668), the human retinoic acid receptor (amino acids 88–153).The highly conserved amino acids of the DNA-binding domain of members ofthe superfamily are as follows:

(SEQ ID No 1) Cys - X - X - Cys - X - X - Asp* - X - Ala* - X - Gly* -X - Tyr* - X - X - X - X - Cys - X - X - Cys - Lys* - X - Phe - Phe -X - Arg* - X - X - X - X - X - X - X - X - X - (X - X -) Cys - X - X -X - X - X - (X - X - X -) Cys - X - X - X - Lys - X - X - Arg - X - X -Cys - X - X -, Cys - Arg* - X - X - Lys* - Cys - X - X - X - Gly* - Met;wherein X designates non-conserved amino acids within the DNA-bindingdomain; the amino acid residues denoted with an asterisk are residuesthat are almost universally conserved, but for which variations havebeen found in some identified hormone receptors; and the residuesenclosed in parenthesis are optional residues (thus, the DNA-bindingdomain is a minimum of 66 amino acids in length, but can contain severaladditional residues).

Exemplary members of the steroid/thyroid superfamily of receptorsinclude steroid receptors such as glucocorticoid receptor,mineralocorticoid receptor, progesterone receptor, androgen receptor,vitamin D₃ receptor, and the like; plus retinoid receptors, such asRARα, RARβ, RARγ, and the like, plus RXRα, RXRβ, RXRγ, and the like;thyroid receptors, such as TRα, TRβ, and the like; as well as other geneproducts which, by their structure and properties, are considered to bemembers of, the superfamily, as defined hereinabove. Examples of orphanreceptors include HNF4 [see, for example, Sladek et al., in Genes &Development 4: 2353–2365 (1990)], the COUP family of receptors [see, forexample, Miyajima et al., in Nucleic Acids Research 16: 11057–11074(1988), Wang et al., in Nature 340: 163–166 (1989)], COUP-like receptorsand COUP homologs, such as those described by Mlodzik et al., in Cell60: 211–224 (1990) and Ladias et al., in Science 251: 561–565 (1991),the ultraspiracle receptor [see, for example, Oro et al., in Nature 347:298–301 (1990)], and the like.

Processes capable of being modulated by retinoid receptors, inaccordance with the present invention, include in vitro cellulardifferentiation and proliferation, in vitro proliferation of melanomacell lines, in vitro differentiation of mouse teratocarcinoma cells (F9cells), in vitro differentiation of human epidermal keratinocytes, limbmorphogenesis, regulation of cellular retinol binding protein (CRBP),and the like. As readily recognized by those of skill in the art, theavailability of ligands for the retinoid X receptor makes it possible,for the first time, to carry out assays for the identification ofantagonists for said receptor.

Processes capable of being modulated by retinoid receptors, inaccordance with the present invention, also include the in vivomodulation of lipid metabolism, in vivo modulation of skin-relatedprocesses (e.g., acne, aging, wrinkling, skin cancer, and the like), invivo modulation of malignant cell development, such as occurs, forexample, in acute promyelocytic leukemia, testicular cancer, lungcancer, and the like. The ability of compounds of the invention tomodulate such processes is evidenced in a number of ways. See, forexample, FIG. 6 where the ability of RXR-alpha, in the presence ofligand therefor (e.g., 9-cis-retinoic acid) is shown to exert a strongeffect on the expression of genes under the control of regulatoryelements of apolipoprotein AI. Similarly, studies with model systems fora variety of disease states (e.g., differentiation of HL60 cells as amodel for acute promyelocytic leukemia, proliferation of melanoma celllines as a model for skin cancer, differentiation of keratinocytes as amodel for non-malignant skin disorders, and the like), as set forth inthe Examples, demonstrate the ability of retinoid receptors, in thepresence of ligand therefor, e.g., 9-cis-retinoic acid, to exert astrong effect on such disease states. Such in vivo applications of theinvention process may allow the modulation of various biologicalprocesses with reduced occurrence of undesirable side effects, and thelike.

In vivo applications of the invention process(es) (and compositions) canbe employed with a wide range of subjects, such as, for example, humans,rodents, sheep, pigs, cows, and the like.

As employed herein, the term “alkyl”, refers to “lower alkyl”, i.e.,alkyl moieties having in the range of 1 up to about 4 carbon atoms,i.e., methyl groups, ethyl groups, propyl groups, isopropyl groups,normal-butyl groups, isobutyl groups, sec-butyl groups, tert-butylgroups, and the like.

Cyclic moieties contemplated as part of the compounds employed in thepractice of the present invention include 5-, 6-, and 7-memberedcarbocyclic, heterocyclic aromatic or heteroaromatic rings. Included inthis definition, for example, are optionally substituted saturated,mono-unsaturated or polyunsaturated carbocyclic species, such as, forexample, cyclopentane, cyclopentene, cyclohexane, cyclohex-2-ene,cyclohex-3-ene, cyclohex-4-ene, and cyclohex-5-ene isomers, and 2,4-,2,5-, and 3,5-cyclohexadiene variants thereof. Examples of heterocyclicspecies contemplated as part of the compounds employed in the practiceof the present invention include dihydrofuran, tetrahydrofuran,dihydrothiophene, tetrahydrothiophene, dihydropyran, tetrahydropyran,dihydrothiopyran, tetrahydrothiopyran, piperidine, pyrrolidine, and thelike, as well as derivatives thereof. Examples of aromatic orheteroaromatic species contemplated as part of the rexoid compounds ofthe present invention include phenyl, tolyl, xylyl, mesityl, benzyl,pyridyl, thiophenyl, furanyl, and the like, as well as derivativesthereof.

Preferred cyclic moieties are typically geminally di-substituted,mono-unsaturated species. Presently preferred geminally di-substituted,mono-unsaturated cyclic moieties are the 1,1,5-trisubstitutedcyclohex-5-ene structure of naturally occurring retinoic acid (i.e., thering structure of β-ionone; the position of the substituents on the ringare designated employing the traditional retinoic acid numberingconvention for the ring structure of β-ionone), as well as the1,1,4,5-tri-substituted cyclohex-5-ene structure provided by hydroxy- orketo-substituted derivatives of the traditional β-ionone structure.

Compounds contemplated for use in the practice of the present inventioninclude compounds having the structure:

wherein:

unsaturation between carbon atoms C⁹ and C¹⁰ has a cis configuration,and one or both sites of unsaturation between carbon atoms C¹¹ throughC¹⁴ optionally have a cis configuration;

“Ring” is a cyclic moiety;

Z is selected from carboxyl, carboxaldehyde, hydroxyalkyl, thioalkyl,hydroxyalkyl phosphate, alkyl ether of a hydroxyalkyl group, alkylthioether of a thioalkyl group, esters of hydroxyalkyl groups,thioesters of hydroxyalkyl group, esters of thioalkyl groups, thioestersof thioalkyl groups, aminoalkyl, N-acyl aminoalkyl, carbamate, and thelike; and

R on each of C⁷,C⁸, C⁹, C¹⁰, ¹¹, C¹², C¹³, or C¹⁴ is independentlyselected from H, halogen, alkyl, aryl, hydroxy, thiol, alkoxy,thioalkoxy, amino, or any of the Z substituents; or

any two or more of the R groups can be linked to one another to form oneor more ring structures.

Presently preferred compounds which are contemplated by the abovegeneric structure include 9-cis-retinoic acid, as well as novelderivatives thereof such as-9-phenyl-9-cis-retinoic acid,4-hydroxy-9-cis-retinoic acid, 4-keto-9-cis-retinoic acid, and the like.

In another preferred embodiment of the present invention, thesubstituents on C⁹ and C¹³ are methyl; in yet another preferredembodiment, the substituents on two or more of the side chain carbons(i.e., C⁷, C⁸, C⁹, C¹⁰, C¹¹, C¹², C¹³, or C¹⁴) can be linked together toform a ring structure. For example, the substituents on C⁸ and C¹¹ canbe linked together to form a structure having a constrained 9-cis doublebond (i.e., a 9-cis locked rexoid derivative), as follows:

wherein:

X is —[(CR₂)_(x)—X′—(CR₂)_(y)]—,

X′ is selected from —O—, carbonyl (>CO), —S—, —S(O)—, —S(O)₂—,thiocarbonyl (>CS), —NR″—, or —CR₂—,

R, Ring and Z are as defined above,

R″ is hydrogen, alkyl, hydroxy, thiol, or alkoxy acyl (—CO—O-alkyl);

x is 0, 1 or 2,

y is 0, 1, or 2, and

x+y≦2.

Such compounds include cyclopentene derivatives, cyclohexenederivatives, cycloheptene derivatives, dihydrofuran derivatives,dihydropyrrole derivatives, and the like, wherein the cyclic structurelinking C⁸ and C¹¹ serves to prevent isomerization of the cis doublebond between C⁹ and C¹⁰.

Especially preferred derivatives of structure I are those where Z is acarboxyl group, and Ring is a β-ionone-like species having thestructure:

wherein:

each R is independently defined as provided above;

any one of C², C³, or C⁴ can be replaced with —O—, carbonyl (>CO), —S—,—S(O)—, —S(O)₂—, thiocarbonyl (>CS), or —NR″—; wherein R″ is as definedabove; and

said cyclic moiety exists as the saturated, 2-ene, 3-ene, 4-ene, or5-ene mono-unsaturated isomer; the 2,4-, 2,5-, or 3,5-diene derivativethereof; or an aromatic derivative thereof.

Especially preferred species for use in the practice of the presentinvention are derivatives of structure I where Z is a carboxyl group,and Ring is a 1,1,5-trisubstituted cyclohex-5-ene structure or a1,1,4,5-tetrasubstituted cyclohex-5-ene structure.

Similarly, the substituents on C¹⁰ and C¹³ can be linked together toform a structure having a constrained 9,11-di-cis configuration (i.e., a9-cis locked rexoid derivative), as follows:

wherein:

X, X′, R, R″, Z, Ring, x and y are as defined above.

Such compounds include cyclopentene derivatives, cyclohexenederivatives, cycloheptene derivatives, dihydrofuran derivatives,dihydropyrrole derivatives, and the like, wherein the cyclic structurelinking C¹⁰ and C¹³ serves to hinder isomerization of the cis doublebond between C⁹ and C¹⁰, and prevent isomerization of the cis doublebond between C¹¹ and C¹².

Especially preferred derivatives of Structure II are those where Z is acarboxyl group, and the Ring is a 1,1,5-trisubstituted cyclohex-5-enestructure or a 1,1,4,5-tetrasubstituted cyclohex-5-ene structure.

Similarly, at least two of the substituents on C⁸, C¹¹, and/or C¹⁴ canbe linked together to form a structure having a constrained 9,13-di-cisconfiguration (i.e., a 9-cis locked rexoid derivative), shown below asStructure III:

wherein:

one A is X and the other A is X′, and

X, X′, R, R″, Z, Ring, x and y are as defined above. Those of skill inthe art recognize that the junction between the two bridging groups (A)can only occur through an atom with a valence of three or four (i.e.,through carbon or nitrogen), so as to accomodate the bonds required tolink the fused rings together.

Similarly, at least two of the substituents on C⁸, C¹¹, and/or C¹⁴ canbe linked together, and further linked to C⁵ of Ring, or to asubstituent on C⁵ to form a structure having a constrained 9,13-di-cisconfiguration (i.e., a 9-cis locked rexoid derivative), shown below asStructure IV:

wherein:

one A is X and the other A is X′,

B is X′, and

X, X′, R, R″, Z, Ring, x and y are as defined above. As noted above withrespect to Structure III, those of skill in the art recognize that thejunction(s) between the bridging groups (A) and (B) can only occurthrough an atom with a valence of three or four (i.e., through carbon ornitrogen), so as to accomodate the bonds required to link the fusedrings together.

Such compounds include cyclopentene derivatives, cyclohexenederivatives, cycloheptene derivatives, dihydrofuran derivatives,dihydropyrrole derivatives, and the like, wherein the cyclic structureslinking C⁸, C¹¹ and/or C¹³ serves to prevent isomerization of the cisdouble bonds at carbon 9 and carbon 13.

Especially preferred derivatives of Structures III and IV are thosewhere Z is a carboxyl group, and Ring is a 1,1,5-trisubstitutedcyclohex-5-ene structure or a 1,1,4,5-tetrasubstituted cyclohex-5-enestructure.

Similarly, the substituents on C¹⁰ and C¹¹ can be linked together toform a structure having a constrained 9-cis double bond (i.e., a 9-cislocked rexoid derivative), as follows:

wherein:

X″ is —[(CR₂)_(a)—X′—(CR₂)_(b)]—,

X′, R, R″, Ring and Z are as defined above,

a is 0, 1, 2, 3 or 4,

b is 0, 1, 2, 3, or 4, and

a+b is ≧2, but ≦4.

Such compounds include cyclopentene derivatives, cyclohexenederivatives, cycloheptene derivatives, dihydrofuran derivatives,dihydropyrrole derivatives, and the like, wherein the cyclic structurelinking C¹⁰ and C¹¹ serves to prevent isomerization of the cis doublebond between C⁹ and C¹⁰.

Especially preferred derivatives of Structure V are those where Z is acarboxyl group, and Ring is a 1,1,5-trisubstituted cyclohex-5-enestructure or a 1,1,4,5-tetrasubstituted cyclohex-5-ene structure.

Similarly, the substituents on C⁷ and C⁹ can be linked together, and thesubstituents on C¹⁰ and C¹² can be linked together to form a structurehaving a constrained 9-cis double bond (i.e., a 9-cis locked rexoidderivative), as follows:

wherein:

Y is —[(CR₂)_(c)—X′—(CR₂)_(d)]—,

X′, R, R″, Ring and Z are as defined above,

c is 0, 1, 2 or 3,

d is 0, 1, 2 or 3, and

c+d≧1 but ≦3.

Such compounds include cyclopentene derivatives, cyclohexenederivatives, cycloheptene, derivatives, dihydrofuran derivatives,dihydropyrrole derivatives, and the like, wherein the cyclic structureslinking C⁷ and C⁹ and C¹⁰ and C¹² serve to prevent isomerization of thecis double bond between C⁹ and C¹⁰.

Especially preferred derivatives of Structure VI are those where Z is acarboxyl group, and Ring is a 1,1,5-trisubstituted cyclohex-5-enestructure or a 1,1,4,5-tetrasubstituted cyclohex-5-ene structure.

Similarly, the substituents on C⁹ and C¹⁰ can be linked together to forma structure having a constrained C-9 double bond (i.e., a 9-cis lockedrexoid derivative), as follows:

wherein:

X″′ is X″ or an unsaturated linking group having the structure:—[Q═CR—J]—,

wherein Q is —N═ or —CR═, and J is —CR═CR—, —N═CR—, —CR═N—, —O—, —S—, or—NR″—,

thereby incorporating C⁹ and C¹⁰ of the rexoid compound into an aromatic(or pseudo-aromatic) ring, and

X′, X″, R, R″, Ring, Z, a and b are as defined above.

Such compounds include cyclohexene derivatives, cycloheptenederivatives, benzene derivatives, pyridine derivatives, furanderivatives, thiophene derivatives, pyrrole derivatives, oxazole,derivatives, thiazole derivatives, imidazole derivatives, pyrazolederivatives, and the like, wherein the cyclic structure linking C⁹ andC¹⁰ serves to prevent isomerization of the C⁹–C¹⁰ double bond; however,rotation about the 8–9 and/or 10–11 single bonds can still occur.

Especially preferred derivatives of Structure VII are those where Z is acarboxyl group, and Ring is a 1,1,5-trisubstituted cyclohex-5-enestructure or a 1,1,4,5-tetrasubstituted cyclohex-5-ene structure.

In addition to the structures set forth above, those of skill in the artcan readily identify additional means to constrain the basiccis-configuration containing rexoid compounds employed in the practiceof the present invention.

In accordance with a preferred embodiment of the present invention, thecyclic moiety has the β-ionone structure set forth above. Especiallypreferred are the 1,1,5-trisubstituted cyclohex-5-ene structure(characteristic of β-ionone) as well as the closely related1,1,4,5-tetrasubstituted cyclohex-5-ene structure from which many rexoidcompounds according to the present invention can be prepared.

In accordance with a particularly preferred embodiment of the presentinvention, the compounds employed in the invention process are selectedfrom 9-cis-retinoic acid and derivatives thereof as contemplated byStructure A set forth above, as well as 9-cis-locked derivatives ofretinoic acid as set forth in Structures I–VII above. Examples ofspecific compounds contemplated for use in the practice of the presentinvention are compounds wherein Z is carboxy, Ring is the1,1,5-trisubstituted cyclohex-5-ene structure characteristic of β-ionone(or the closely related 1,1,4,5-tetrasubstituted cyclohex-5-ene), andhaving a side chain structure(s) as described above for StructuresI–VII.

“Rexoid” derivatives as described above can be prepared employing avariety of synthetic methods, which are readily available (and wellknown) to those of skill in the art. See, for example, the methodsdescribed in Chemistry and Biology of Synthetic Retinoids, Dawson andOkamura, eds., CRC Press, Inc. (1990), especially Chapter 4, by Ito(found at pages 78–97), and Chapter 9, by de Lera et al. (found at pages202–227) can readily be adapted for the preparation of the compoundsdescribed herein. The contents of this publication are herebyincorporated by reference herein. See also Asato et al., J. Am. Chem.Soc. 108: 5032 (1986); Sheves et al., J. Am. Chem. Soc. 108: 6440(1986); Akita et al., J. Am. Chem. Soc. 102: 6370 (1980); Derguini andNakanishi, Photobiochem. and Photobiophys. 13: 259 (1986), the entirecontents of each of which is hereby incorporated by reference herein.

In accordance with another embodiment of the present invention, there isprovided a method for modulating processes mediated by retinoidreceptors, said method comprising conducting said process in thepresence of:

(a) at least one compound of the structure:

wherein:

each site of unsaturation in the side chain comprising carbon atoms C⁷through C¹⁴ has a trans configuration;

“Ring”, Z, and R are as previously described, and

(b) a cis/trans isomerase capable of converting at least the 9-doublebond from the trans configuration to the cis-configuration.

As employed herein, the term “cis/trans isomerase” refers to enzymeswhich promote a change of geometrical configuration at a double bond.Examples of such enzymes include maleate isomerase, maleylacetoacetateisomerase, retinal isomerase, maleylpyruvate isomerase, linoleateisomerase, furylfuramide isomerase, and the like.

In accordance with yet another embodiment of the present invention,there is provided a method to produce compound(s) of the structure:

wherein:

unsaturation between carbon atoms C⁹ and C¹⁰ has a cis configuration,and one or both sites of unsaturation between carbon atoms C¹¹ throughC¹⁴ optionally have a cis configuration;

“Ring” is a cyclic moiety;

Z is selected from carboxyl, carboxaldehyde, hydroxyalkyl, thioalkyl,hydroxyalkyl phosphate, alkyl ether of a hydroxyalkyl group, alkylthioether of a thioalkyl group, esters of hydroxyalkyl groups,thioesters of hydroxyalkyl group, esters of thioalkyl groups, thioestersof thioalkyl groups, aminoalkyl, N-acyl aminoalkyl, carbamate, and thelike; and

each R is independently selected from H, halogen, alkyl, aryl, hydroxy,thiol, alkoxy, thioalkoxy, amino, or any of the Z substituents;

from the corresponding all-trans configuration material, said methodcomprising contacting said all-trans configuration material with acis/trans isomerase under isomerization conditions.

In accordance with still another embodiment of the present invention,there are provided novel compositions comprising compound(s) ofStructure A (excluding previously identified compounds such as retinoicacid as well as constrained compounds selected from Structures I–VII, asset forth above. Examples of such compounds include9-phenyl-9-cis-retinoic acid, 4-hydroxy-9-cis-retinoic acid,4-keto-9-cis-retinoic acid, and the like. Presently preferred compoundsare those wherein Z is carboxyl and Ring is a 1,1,5-trisubstitutedcyclohex-5-ene structure or a 1,1,4,5-tetrasubstituted cyclohex-5-enestructure.

The invention compounds can be employed for both in vitro and in vivoapplications. For in vivo applications, the invention compounds can beincorporated into a pharmaceutically acceptable formulation foradministration. Those of skill in the art can readily determine suitabledosage levels when the invention compounds are so used.

As employed herein, the phrase “suitable dosage levels” refers to levelsof compound sufficient to provide circulating concentrations high enoughto effect activation of retinoid receptor(s). Such a concentrationtypically falls in the range of about 10 nM up to 2 μM; withconcentrations in the range of about 100 nM up to 200 nM beingpreferred.

In accordance with a particular embodiment of the present invention,compositions comprising at least one 9-cis-retinoic acid-like compound(as described above), and a pharmaceutically acceptable carrier arecontemplated. Exemplary pharmaceutically acceptable carriers includecarriers suitable for oral, intravenous, subcutaneous, intramuscular,intracutaneous, and the like administration. Administration in the formof creams, lotions, tablets, dispersible powders, granules, syrups,elixirs, sterile aqueous or non-aqueous solutions, suspensions oremulsions, and the like, is contemplated.

For the preparation of oral liquids, suitable carriers includeemulsions, solutions, suspensions, syrups, and the like, optionallycontaining additives such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring and perfuming agents, and the like.

For the preparation of fluids for parenteral administration, suitablecarriers include sterile aqueous or non-aqueous solutions, suspensions,or emulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized, for example,by filtration through a bacteria-retaining filter, by incorporatingsterilizing agents into the compositions, by irradiating thecompositions, or by heating the compositions. They can also bemanufactured in the form of sterile water, or some other sterileinjectable medium immediately before use.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLES Example 1 Identification of Compound(s) that Activate RXR

In order to a certain if retinoic acid can be converted to a productthat binds directly to RXR, thereby resulting in modulation oftranscription, a strategy was developed to identify retinoic acidmetabolites that might modulate the transcriptional properties of RXR.The identification of any such active metabolite would allow one tofurther determine whether this metabolite was capable of directlybinding to the receptor protein.

Accordingly, the Drosophila melanogaster Schneider cell line (S2) wasincubated with or without all-trans-retinoic acid (RA) for a period of24 hours. Prior to the addition of retinoic acid, Drosophilamelanogaster Schneider cell line (S2) cells were grown in SchneiderDrosophila medium (GIBCO) supplemented with penicillin, streptomycin and12% heat inactivated FCS (Irvine Scientific). One hundred tissue cultureflasks (75 cm²) were set up with 10⁷ cells and 12 ml of medium/flask.Twenty four hours later, either all-trans-retinoic acid (or ethanolsolvent control) was added to each flask to a final concentration of5×10⁻⁶ M in reduced light conditions. Cells were harvested 24 hourslater by centrifugation for 5 minutes at 800 g. Cells were washed twicewith PBS and the resultant pellets were frozen at −80° C. untilextraction.

In parallel, CV-1 cells were set up on 64 tissue culture dishes (150 mm)at 2×10⁶ cells and 25 ml of medium/dish. Cells were treated withretinoic acid and harvested as with the S2 cells except that the CV-1cells (which are adherent) were washed in their dishes with PBS andscraped with a rubber policeman prior to centrifugation and freezing.

Following incubation, the cell pellets were collected, organicallyextracted and chromatographically fractionated by HPLC. The various HPLCfractions were assayed for their ability to produce a ligand dependentincrease in transcriptional activity mediated by RXR. This assay systeminvolves transfecting cells with the cDNA for the RXR receptor and aluciferase reporter molecule which is under control of a promotercontaining a RXR response element (RXRE) [see Mangelsdorf et al., Cell66:555 (1991)]. The addition of a ligand capable of activating RXRresults in an increase in luciferase activity.

Schneider cells, CV-1 cells and mouse tissues were extracted asdescribed by C. Thaller and G. Eichele in Nature Vol. 327:625 (1987).Mouse tissue was used to determine if any RXR ligand is present in vivo.In the case of tissue extractions, 2.10⁵ dpm internal standard[11,12-³H]-all-trans-retinoic acid (New England Nuclear) or9-cis-retinoic acid (generated by isomerization with light) were addedto the homogenate. Extracts were fractionated on a Waters Novapak 300 mmC₁₈ analytical column at a flow rate of 1 ml min⁻¹. The mobile phase (G)was a 1:1 mixture of:

A [CH₃CN/CH₃OH/2% aqueous CH₃COOH (3:1:1)] and

E [CH₃CN/CH₃OH/2% aqueous CH₃COOH (11:3:10)].

Other mobile phases used have the following compositions:

C: CH₃CN/CH₃OH/H₂O/CH₃COOH (80:10:10:1),

H: mix CH₃OH/10 mM ammonium acetate (9:1) with equal volume of CH₃OH/10mM ammonium acetate (3:1).

Methyl esters of retinoic acid isomers and/or metabolites contained inthe HPLC fractions were generated as described in Wedden et al. [(Meth.Enzymol. 190:201 (1990)]. Reference standards used were from Aldrich,Sigma or kindly provided by Hoffmann-LaRoche. Authentic 9-cis-retinol,9-cis-retinoic acid and 9-cis-methylretinoate were either synthesizedfrom 9-cis-retinal (see E. J. Corey et al., J. Am. Chem. Soc. 90:5616(1968); C. D. B. Bridges & R. A. Alvares [(Meth. Enzymol. 81:463 (1982)]or generated by photoisomerization of the all-trans isomer followed byfractionation of the resulting isomers by HPLC.

Photoisomerization of all-trans-retinoic acid is carried out employingstandard isomerization techniques which are well known to those of skillin the art. For example, retinoic acid can be dissolved in a polarorganic solvent such as ethanol, placed in a quartz cuvette, andirradiated with a variety of wavelengths of light (such as fluorescentlight). Temperature at which irradiation is carried out is not critical;accordingly, irradiation can be carried out at room temperature.Irradiation time is also not critical; typical irradiation times are inthe range of about 0.5–2 hours.

The various HPLC fractions were diluted 1:100 and assayed for theirability to modulate the transcriptional properties of RXR.

Cotransfection Assay in CV-1 Cells

A monkey kidney cell line, CV-1, was used in the cis-trans assay. Cellswere transfected with two DNA transfection vectors. The trans-vectorallowed efficient production of retinoid receptor (e.g., RAR or RXR) inthese cells, which do not normally express these receptors. Thecis-vector contains an easily assayable gene, in this case the fireflyluciferase, coupled to a retinoid-responsive promoter. Addition ofretinoic acid or an appropriate synthetic retinoid results in theformation of a retinoid-receptor complex that activates the luciferasegene, causing light to be emitted from cell extracts. The level ofluciferase activity is directly proportional to the effectiveness of theretinoid-receptor complex in activating gene expression. This sensitiveand reproducible cotransfection approach permits the identification ofretinoids that interact with the different receptor isoforms.

Cells were cultured in DMEM supplemented with 10% charcoalresin-stripped fetal bovine serum, and experiments were conducted in96-well plates. The plasmids were transiently transfected by the calciumphosphate method [Umesono and Evans, Cell 57:1139–1146 (1989); Berger etal., J. Steroid Biochem. Molec. Biol. 41:733–738 (1992)] by using 10 ngof a pRS (Rous sarcoma virus promoter) receptor-expression plasmidvector, 50 ng of the reporter luciferase (LUC) plasmid, 50 ng ofPRSβ-GAL (β-galactosidase) as an internal control, and 90 ng of carrierplasmid PGEM. Cells were transfected for 6 hours and then washed toremove the precipitate. The cells were then incubated for 36 hours withor without retinoid. After the transfection, all subsequent steps wereperformed on a Beckman Biomek Automated Workstation. Cell extracts wereprepared as described by Berger et al. supra, then assayed forluciferase and β-galactosidase activities. All determinations wereperformed in triplicate in two independent experiments and werenormalized for transfection efficiency by using β-galactosidase as theinternal control. Retinoid activity was normalized relative to that ofretinoic acid and is expressed as potency (EC50), which is theconcentration of retinoid required to produce 50% of the maximalobserved response, and efficacy (%), which is the maximal responseobserved relative to that of retinoic acid at 10⁻⁵ M.

The receptor expression vectors used in the cotransfection assay havebeen described previously [pRShRAR-α: Giguere et al., Nature 330:624–629(1987); pRShRAR-β and pRShRAR-γ: Ishikawa et al., Mol. Endocrinol.4:837–844 (1990); retinoid X receptor-alpha (RXR-α) [see Mangelsdorf etal., in Nature 345: 224–229 (1990)], retinoid X receptor-beta (RXR-β)and retinoid X receptor-gamma (RXR-γ) [see Mangelsdorf et al., Genes andDevelopment 6:329–344 (1992)]. A basal reporter plasmid ΔMTV-LUC[Hollenberg and Evans, Cell 55:899–906 (1988)] containing two copies ofthe TRE-palindromic response element 5′-TCAGGTCATGACCTGA-3′ [SEQ ID No2; see Umesono et al., Nature 336:262–265 (1988)] was used in alltransfections for the retinoid receptors.

The bacterial expression vector for PET-8c-RAR-α used in the competitivebinding assay has been reported [Yang et al., Proc. Natl. Acad. Sci. USA88:3559–3563 (1991)]. Similar expression vectors employing the PET-8cvector system [Studier et al., Methods in Enzymology 185:60–69 (1990)]were constructed for RAR-β and RAR-γ.

The transactivation profile of RXR-alpha with the various HPLC fractionscontaining various retinoic acid isomers and/or metabolites is shown inFIG. 1. These data reveal two distinct regions of activity, onerelatively early (fraction 7) and a second broader region of activity(fractions 16–21) that elutes considerably later. The all-trans-retinoicacid coelutes in fractions 20 and 21 (FIG. 1) and is the major U.V.absorbing material present in the cell extracts. However, the activityprofile demonstrates that, in addition to all-trans-retinoic acid, thereare active components that must be derived from, or induced by,all-trans-retinoic acid that activate RXR.

To identify potential compounds that would be as effective or moreactive than all-trans-retinoic acid, one must take into account not onlythe activity of the individual fractions, but also their concentrations.All active fractions were therefore reassayed over a broad range ofconcentrations, taking into account the relative concentrations of theindividual fractions. To determine the relative concentrations of thefractions, the following initial assumptions were made: 1) the activefractions are retinoic acid metabolites and 2) the molar extinctioncoefficient of the various active fractions is relatively similar (i.e.,within a factor of two). This assumption is supported by values reportedin the literature for a large number of retinoids. A comparison of thetransactivation profile of all-trans-retinoic acid, (i.e., fraction 20)on RAR-alpha and RXR-alpha is shown in FIG. 2 a. Although the maximalactivation (i.e., efficacy) of RAR and RXR with retinoic acid issimilar, RAR is more sensitive by a factor of approximately 10 fold(i.e., 10 fold more potent). In contrast, analysis of the variousfractions produced as describes above demonstrates that fraction 18 isconsiderably more active on RXR than RAR (see FIG. 2 b). These datasuggest that a metabolic product present in S2 cells pretreated withretinoic acid is a more potent activator of the RXR subfamily than theRAR subfamily.

Example 2 Identification of 9-cis Retinoic Acid as a Transactivator ofRXR

Two observations suggest that fraction 18 (peak X, see FIG. 1) is acellular metabolite of all-trans-retinoic acid. First, extracts ofSchneider cells grown in the absence of all-trans-retinoic acid do notexhibit peak X. Second, when cells are exposed to all-trans-retinoicacid, X appears in a time-dependent fashion.

Therefore, to chemically identify X, fraction 18 was subjected tochemical derivatization, high performance liquid chromatography (HPLC)and gas chromatography/mass spectrometry (GC/MS). It was found that uponmethylation with diazomethane, the retention time of peak X shiftsdramatically (i.e., from 10.2 minutes to 19.5 minutes under the HPLCconditions used). This indicates that the compound(s) corresponding topeak X has a free carboxyl group. When methylated X was analyzed byGC/MS, the electron impact mode revealed that X gives rise to amolecular ion at m/z 314, corresponding to that of a retinoic acidmethyl ester. This suggests that X is a stereoisomer of retinoic acid.To determine which isomer X represents, the retention time of X wascompared with that of 9-cis-, 11-cis- and 13-cis-retinoic acid. It wasfound that X coelutes with authentic 9-cis-retinoic acid. Furthermore,the methyl ester of X coelutes with 9-cis-methylretinoate, and when themethyl ester of X is reduced to the alcohol with lithium aluminumhydride, the resulting product coelutes with authentic 9-cis-retinol.

For GC/MS analysis, methylated retinoic acid isomers were dissolved inhexane. The sample was injected via a falling needle injector (280° C.)into a 30 m×0.32 mm fused silica DB5 capillary column (J+J scientific)inserted directly into the ion source of a VG Trio-1000 massspectrometer operating in electron impact mode (70 eV). The sample waseluted with a temperature gradient (200–300° C., 10° C. min⁻¹).

Finally, the mass spectrum of authentic 9-cis-retinoic acid methyl esterand that of methylated peak X are found to be identical. Taken togetherthese analyses establish that peak X represents 9-cis-retinoic acid.Although earlier work indicated the presence of 9-cis-retinol in fishliver, it was not clear whether 9-cis-retinoic acid existed in vivo(i.e., whether 9-cis-retinoic acid is a physiological compound). To findout if 9-cis-retinoic acid exists in vivo, mouse liver and kidneytissues were extracted. These tissues were selected because they containa broad spectrum of retinoid metabolites and also express RXR. Prior toextraction, radiolabeled 9-cis-retinoic acid was added to the kidneyhomogenate to serve as an internal standard. Extracts were firstfractionated on a reverse phase column (Waters Novo pak 300 mm C₁₈analytical column at a flow rate of 1 ml/min) using mobile phase G.

Fractions from the kidney extracts containing radioactive internalstandard were rechromatographed on a second C₁₈ column using mobilephase H. This procedure gave a small, but distinct absorbance peak whichco-migrated with authentic 9-cis-retinoic acid.

Similarly, liver extract was fractionated on a reverse .phase column andeluted with mobile phase G. However under the conditions employed,9-cis-retinoic acid eluted with all-trans-retinol (which is abundantlypresent in the liver). To separate these two retinoids, this fractionwas methylated with diazomethane and then reanalyzed by HPLC employingmobile phase C. This approach resulted in a distinct peak coeluting withthe authentic methyl ester of 9-cis-retinoic acid.

To rule out the possibility that 9-cis-retinoic acid had formed duringthe extraction procedure from all-trans-retinoic acid, liver tissuehomogenate was spiked with tritiated all-trans-retinoic acid. SubsequentHPLC fractionation revealed that 94% of the radioactivity still residedin all-trans-retinoic acid, approximately 5% in 13-cis-retinoic acid and1% or less in 9-cis-retinoic acid. Based on peak area integration theconcentrations of 9-cis-retinoic acid in liver and kidney are estimatedto be ˜4 ng, and ˜4 ng, respectively, per g of wet weight. Thisindicates that endogenous 9-cis-retinoic acid is not formed fromall-trans-retinoic acid during extraction. In conclusion, theseexperiments establish that 9-cis-retinoic acid is a naturally occurringretinoic acid isomer.

Example 3 Transactivation Profile of Retinoid Isomers on RXR and RAR

The establishment that peak X represents a stereoisomer ofall-trans-retinoic acid suggested that the various retinoid isomers mayhave different retinoid receptor activation profiles. To further analyzethe ability of retinoic acid isomers to modulate the transcriptionalproperties of RXR-alpha and RAR-alpha, the four major photoisomers ofall-trans-retinoic acid were identified and assayed for the ability totransactivate RXR and RAR. FIG. 3 shows the dose response curves for13-cis-, 11-cis-, 9-cis- and all-trans-retinoic acid for both RAR-alphaand RXR-alpha.

Of the four major isomers of retinoic acid, 9-cis-retinoic acid is seento be the most potent and efficacious activator of RXR-alpha in bothinsect S2 cells (see FIG. 3A) and mammalian CV-1 cells (see FIG. 3B).The maximal response (EC50 value) is 10⁻⁸ M and 5×10⁻⁸ M, respectively.The observed rank order of potency for the different isomers is the samein both cell lines. 9-cis-retinoic acid is approximately 40 fold morepotent as an activator of RXR than 11-cis-, 13-cis- orall-trans-retinoic acid. These transactivation data strongly suggestthat 9-cis-retinoic acid is an endogenous RXR-alpha activator.

In contrast, 9-cis-retinoic acid is equipotent to all-trans-retinoicacid as an activator of RAR-alpha (FIG. 3C). The EC50 value for9-cis-retinoic acid on RAR-alpha is 2×10⁻⁷ M. 9-cis-retinoic acid is themost potent RXR-alpha ligand to be tested to date.

Similarly, transactivation of other isoforms of RXR (i.e., RXR-beta,RXR-gamma) and RAR (i.e., RAR-beta, RAR-gamma) by 9-cis-retinoic acidwas also examined. 9-cis-retinoic acid was also found to be a potentactivator of these isoforms as well, as shown in Table 1:

TABLE 1 EC₅₀* (nM) Receptor All-trans-retinoic Acid 9-cis-retinoic AcidPAR-α 3861 ± 13 327 ± 30 RAR-β  152 ± 12  95 ± 13 PAR-γ  48 ± 8   61 ±5  RXR-α 1174 ± 26 255 ± 17 RXR-β 1841 ± 26 218 ± 17 RXR-γ 1369 ± 26 254± 19 *Mean ± SEM

Example 4 9-cis Retinoic Acid Binds Directly to RXRs

The ability of 9-cis-retinoic acid to transactivate RXR-alpha suggestedtesting to see whether 9-cis-retinoic acid was also capable of bindingdirectly to RXRs. RXR-alpha was expressed in baculovirus and was shownto have biochemical properties that were identical to the mammalianexpressed protein. The baculovirus expressed protein had a molecularweight of 51,000, reacted specifically with RXR-alpha antibody and wascapable of binding in vitro to DNA sequences that have been previouslyshown to be specific RXR response elements (i.e. CRBPII, see Mangelsdorfet al., Cell 66:555 (1991); apolipoprotein AI gene, see Rottman et al.,Mol. Cell Biol. 11:3814 (1991)].

To characterize the ligand binding characteristics of 9-cis-retinoicacid to baculovirus-derived RXR, saturation binding analysis was carriedout (see FIG. 4). Radiolabelled 9-cis-retinoic acid binds specificallyto RXR-alpha in a saturable manner. Scatchard analysis suggests a singlehigh affinity binding site with a Kd value of 11.7 nM (see FIG. 4 b).Under identical binding conditions [³H]-all-trans-retinoic acid did notbind to RXR-alpha (see FIG. 4 a). In addition, 9-cis-retinoic acid wasalso capable of binding specifically to RAR-alpha as a high affinityligand. 9-cis-retinoic acid did not bind to mock baculovirus extracts(i.e., control extracts from cells that do not express RXRs).

Similarly, binding studies were also carried out with other isoforms ofRXR (i.e., RXR-beta, RXR-gamma), other isoforms of RAR (i.e., RAR-beta,RAR-gamma), and cellular retinoic acid binding protein (CRABP) withall-trans-retinoic acid and 9-cis-retinoic acid. Whileall-trans-retinoic acid is known to bind to each of these “receptors”,9-cis-retinoic acid was also found to bind to the other isoforms ofretinoid receptors (but not to the cellular retinoic acid bindingprotein, CRABP), as shown in Table 2:

TABLE 2 Kd (nM) Receptor All-trans-retinoic Acid 9-cis-retinoic AcidPAR-α 0.4 0.3 PAR-β 0.4 0.2 RAR-γ 0.2 0.8 RXR-α No binding 1.5 RXR-β Nobinding 2.1 RXR-γ No binding 1.9 CRABP 20 >100

The properties of many members of the steroid hormone receptorsuperfamily have been characterized and defined using DNA cellulosechromatography [see, for example, Pike and Haussler, Proc. Natl. Acad.Sci. USA 76:5485 (1979) and Pike et al., J. Biol. Chem. 258:1289(1983)]. Receptors, such as the VDR, have been shown in the presence oftheir cognate ligand to bind to DNA-cellulose [see, for example,Allegretto et al., J. Biol. Chem. 262:1312 (1987)] with high affinityand the ligand-receptor complex elutes with a salt gradient. ADNA-cellulose column profile of the baculovirus expressed RXR that hadbeen prelabeled with [³H]-9-cis-retinoic acid is shown in FIG. 5. Thetwo different profiles represent 1) the total amount of[³H]-9-cis-retinoic acid bound and 2) the level of binding that remainsin the presence of 200-fold excess of cold (i.e. non-labeled9-cis-retinoic acid).

There is a peak of radioactivity (marked in the Figure by an arrow) thatelutes off the DNA-cellulose column at 0.15 M KCl. This elution profileis similar to that seen with RARα in the presence of[³H]-all-trans-retinoic acid. A 200 fold excess of cold ligand (i.e.non-specific) is capable of competing greater than 90% of the totalradioactivity bound, demonstrating that the radioactivity in the peakfractions is 9-cis-retinoic acid specifically bound to RXR.

The radioactivity eluted off the column was extracted with organicsolvent and subjected to HPLC analysis.

Inspection of FIG. 5 b makes it clear that the radioactivity bound toRXR co-chromatographs with authentic 9-cis-retinoic acid. Thisobservation further confirms that [³H]-9-cis-retinoic acid is thespecies bound to RXR.

To demonstrate that the protein contained in the peak fractions isindeed RXR, these fractions (labelled 1–15 in FIG. 5 a) were subjectedto immunoblot analysis using an RXRα specific polyclonal antiserum (seeFIG. 5 a, top). All fractions containing radioactivity display adistinct RXRα band at a M_(r) of 51,000. When a similar experiment wasconducted with a baculovirus mock extract, no specific radioactivity wasretained on the column. Taken together, these data strongly suggest that9-cis-retinoic acid is capable of binding specifically to RXR.

Protein samples were resuspended in 2× sample buffer [Laemelli, NatureVol. 227:680(1970)] and boiled for 5 minutes prior to loading onto a 9%SDS polyacrylamide gel. After electrophoretic separation the gels wereelectroblotted onto nitrocellulose membranes (Scheicher and Schuell) for8 hours at 30 volts using a Hoeffer electro-transfer apparatus.Membranes were then incubated in 10% isopropanol, 10% acetic acid for 15minutes, washed 5 minutes in deionized H₂O and 5 minutes in T-TBS buffer(10 mM Tris pH 7.5, 150 mM NaCl and 0.5% Triton X-100). The membraneswere blocked in 5% nonfat milk in T-TBS for 1 hour. The remainder of theprotocol was adapted from the Amersham ECL (Enhanced Chemiluminescence)Western blotting detection system kit. The primary antibody was a rabbitpolyclonal serum raised against a synthetic peptide corresponding toamino acids 214–229 of hRXRα[Kliewer et al., Proc. Natl. Acad. Sci. USA89:1448–1452 (1992)]. The primary antiserum was diluted 1:5000 in T-TBS.The secondary antibody (Donkey anti rabbit IgG conjugated to horseradishperoxidase, Amersham) was used at a dilution of 1:2500.

Example 5 Effects of Topical Application of 9-cis-retinoic Acid(Compared with All-trans-retinoic Acid) on Horn-filled Utriculus Size inthe Rhino Mouse

All-trans-retinoic acid is known to influence cell differentiation andexert profound therapeutic benefits in the treatment of keratinizationdisorders [Elias et al., Arch. Dermatol. Vol. 117:160–180 (1981)].Mezick et al. [see J. Invest. Derm. Vol. 83:110–113 (1984)] demonstratedthat topical treatment of rhino mice (hr hr) with all-trans-retinoicacid could reduce keratinized pilosebaceous structures (horn-filledutriculus). This animal test model was used to evaluate the“antikeratinizing” effects of 9-cis-retinoic acid. Results aresummarized in Table 3:

TABLE 3 Pilosebaceous structure size (% red′n) Vehicle Control 178 μm9-cis-retinoic acid, 0.1%  52 μm (−74%) 0.01%  72 μm (−64%)All-trans-retinoic acid, 0.1%  44 μm (−78%) 0.01%  50 μm (−75%)

9-cis-retinoic acid reduced the mean utriculi diameter after 14 days oftopical application. These results demonstrate that topical applicationof. 9-cis-retinoic acid over a 14 day period can reduce keratinizedpilosebaceous structures (horn-filled utriculus) in Rhino mouse skin.Reduction in the mean utriculi diameter by 9-cis-retinoic acid wascomparable to that observed with all-trans-retinoic acid.

Example 6 Effects of 9-cis-retinoic Acid (Compared withAll-trans-retinoic Acid) on Differentiation of HL60 Cells

Retinoids are known to differentiate human promyelocytic leukemia cells.Differentiation of HL60 cells (a model system for promyelocyticleukemia) can be assessed by Nitro Blue Tetrazolium (NBT) dye reduction(superoxide anion generation) and by measurement of up-regulation of thegene encoding the β subunit of the leukocyte adherence receptor, CD18(J. B. C. vol. 263 No. 27, pp. 13863–13867).

The EC-50 for 9-cis-retinoic acid-mediated differentiation, asdetermined by NBT after 6 days treatment, was 0.2 μM compared to 2 μMfor all-trans-retinoic acid. Maximal effects (efficacies) werecomparable, and CD18 was up-regulated by both ligands. Alpha-interferonpotentiated both all-trans-retinoic acid and 9-cis-retinoicacid-mediated differentiation, as determined by NBT.

HL60R cells have been shown to be resistant to differentiation byall-trans-retinoic acid, probably related to a mutation in the retinoicacid receptor-alpha gene. This cell line was found to be resistant todifferentiation (NBT) by both all-trans-retinoic acid and 9-cis-retinoicacid at concentrations up to 10 μM.

9-cis-retinoic acid effects differentiation of HL60 cells as evidencedby NBT and up-regulation of CD18. Compared with all-trans retinoic acid,9-cis retinoic acid is more potent with similar efficacy.

Example 7 Effects of 9-cis-retinoic Acid (Compared withAll-trans-retinoic Acid) on In vitro Proliferation of Melanoma CellLines

All-trans-retinoic acid and several synthetic analogs (retinoids) havebeen shown to prevent the development of benign and malignant,chemically induced epithelial tumors in vivo [Sporn et al., Fed. Proc.Vol. 35:1332–1338 (1976)]. Lotan et al. (J. Natl. Cancer, Vol.60:1035–1041, 1978) found that all-trans-retinoic acid inhibited thegrowth of several tumor cell lines in vitro. In view of these earlierfindings, it was of interest to evaluate the growth inhibitoryproperties of 9-cis-retinoic acid.

9-cis-retinoic acid inhibited the growth of the murine melanoma cellline Clone M3 in a concentration dependent manner, as follows:

% Growth inhibition (Conc added) 1 μM 0.01 μM 9-cis-retinoic acid −85%−49% all-trans-retinoic acid −94% −48%

Similarly, 9-cis retinoic acid inhibited the growth of the human primarymetastatic melanoma cell line c81–46c in a concentration dependentmanner.

% Growth inhibition (Conc added) 1 μM 0.01 μM 9-cis-retinoic acid −45%−28% all-trans-retinoic acid −44% −17%

In summary, 9-cis-retinoic acid has been shown to inhibit the in vitroproliferation of murine melanoma cell line Clone M3 and human metastaticmelanoma cell line c81–46c in a concentration dependent manner.9-cis-retinoic acid has an equal inhibitory effect on these cells ascompared to all-trans-retinoic acid.

Example 8 Effects of 9-cis-retinoic Acid (Compared withAll-trans-retinoic Acid) on Differentiation of F9 Cells

Retinoids are known to differentiate mouse teratocarcinoma cells (F9).Differentiation of F9 cells is specifically associated with irreversiblechanges in morphology and induction of the biochemical marker alkalinephosphatase (ALP) and tissue plasminogen activator (tPA) (Biochem. J.Vol. 274:673–678).

Both all-trans-retinoic acid and 9-cis-retinoic acid induceddifferentiation of F9 cells into partial endoderm-like cells asindicated by irreversible changes in cellular morphology.All-trans-retinoic acid was 40 times more potent than 9-cis-retinoicacid in inducing ALP, maximal responses were similar.

The response of tissue plasminogen activator factor was less for9-cis-retinoic acid than for all-trans-retinoic acid. At a concentrationof 1 μM of 9-cis-retinoic acid (or all-trans-retinoic acid), increasedcellular activities of tPA by 0.48±0.05 and 0.80±0.08, respectively wereobserved. This effect was concentration-dependent.

In summary, 9-cis-retinoic acid promoted differentiation of F9 cells asevidenced by changes in morphology and marker enzyme activities.Compared with all-trans-retinoic acid, 9-cis-retinoic acid was lesspotent with regard to both enzyme markers. Efficacy was comparable withALP but indeterminate for tPA.

Example 9 Effects of 9-cis-retinoic Acid (Compared withAll-trans-retinoic Acid) on Differentiation of Keratinocytes

Retinoids are known to inhibit squamous cell differentiation of culturednormal human epidermal keratinocytes (NHEK534 cell line), as judged bymorphological alterations and inhibition of induction oftransglutaminase (Type I) (J. Biol. Chem. Vol. 261:15097, 1986; Lab.Invest. Vol. 56:654, 1987).

Both all-trans-retinoic acid and 9-cis-retinoic acid inhibited squamouscell differentiation in a concentration dependent manner as judged bymorphological changes and by transglutaminase activity. The EC50s forinhibition of differentiation by all-trans-retinoic acid and9-cis-retinoic acid were identical (20±2.8 nM). 9-cis retinoic acid andall-trans-retinoic acid EC50s and potencies were nearly identical foreffects on transglutaminase activities.

In summary, like all-trans-retinoic acid, 9-cis-retinoic acid inhibitsmorphological differentiation of NHEK534 cells and induction oftransglutaminase activity.

Example 10 Synthesis of 9-Phenyl-9-cis-retinoic Acid

To a solution of 44 mg (0.10 mmole) of the following phosphonatereagent:

in THF (0.5 ml) at room temperature was added NaH (60% in oil, 5 mg;0.13 mmole) and the mixture stirred at that temperature for 10 minutes.To this, 26 mg (0.08 mmole) of the aldehyde:

in THF (0.5 ml) was added at room temperature and the mixture allowed tostir for 30 minutes. Aqueous workup in the usual manner (NH₄Cl (aq),H₂O, brine, MgSO₄) gave a mixture of 9-phenyl-9-cis ester and9-phenyl-9,13-dicis ester (30 mg, 92%) (the calculated ratio of 9-cis:9,13-dicis=4:1).

Ethyl Ester of 9-phenyl-9-cis-retinoic Acid:

Ethyl Ester of 9-phenyl-9,13-dicis-retinoic Acid:

To a mixture of 9-cis and 9,13-dicis ester (20 mg, 0.05 mmole) inmethanol (0.7 ml) and H₂O (0.7 ml) at 25° C. was added KOH (14.3 mg,0.25 mmole). Consequently, the mixture was heated to 70° C. for 2 hours.The reaction was then cooled down to 0° C., diluted with 10 ml ofdiethyl ether), and acidified with HCl (0.12M in HCl, 2.17 ml). Aqueousworkup in the usual manner (H₂O, brine, MgSO₄) gave a mixture of 9-cisand 9,13-dicis acid. Flash column chromatography (silica, 13% ethylacetate in benzene) gave pure 9-phenyl-9-cis retinoic acid (14.5 mg.100%).

The ¹HNMR spectrum of 9-phenyl-9-cis retinoic acid is as follows:

¹HNMR (400 mHz), CDCl₃) δ 7.4–7.3 (m, 5H, aromatic), 7.20 (dd, J=16, 12Hz, 1H, olefinic), 6.60 (d, J=16 Hz, 1 H, olefinic), 6.38 (d, J=16 Hz, 1H, olefinic), 6.25 (d, J=12 Hz, 1H, olefinic), 6.15 (d, J=16 Hz, 1 H,olefinic), 5.80 (s. 1H, olefinic), 2.48 (s. 3H, CH₃l, 2.05 (t. J=5Hz,2H, CH₂), 1.79 (s, 3H, CH₃), 1.70–1.40 (m, 4H, CH₂—CH₂), 1.00 (s, 6H,2×CH₃)9-phenyl-9-cis RA: TLC Rf 0.23 (13% ethyl acetate in Benzene)

Example 11 Synthesis of 4-hydroxy-9-cis-retinoic Acid

To a solution of 9-cis-retinoic acid (51 mg, 0.17 mmole) in 1.4-dioxane(2 ml) was added SeO₂ (19 mg, 0.17 mmole) at 60° C. The solution wasallowed to stir at that temperature for 3 hours. The reaction mixturewas then filtered through a silica bed. The filtrate was concentratedand the residue subjected to flash column chromatography (silica, 75%ether in petroleum ether) to afford 4-OH-9-cis-retinoic acid (21 mg.,40% yield), which is characterized as follows: Oil; TLC Rf=0.25 (silica,75% ether in petroleum ether); ¹HNMR (400 MHz, CDCl₃) δ 7.08 (dd, J=16,12 Hz, 1H, olefinic); 6.64 (d, J=16 Hz, 1H, olefinic), 6.21 (d, J=16 HZ,1H, olefinic), 6.20 (d, J=16 Hz, 1H, olefinic); 6.04 (d, J=12 Hz,olefinic), 5.79 (s, 1H, olefinic), 4.02 (t, J=5 Hz, 1H, CH—O), 2.18 (s,3H, CH₃), 2.02 (s, 3H, CH₃), 1.82 (s, 3H, CH₃), 2.0–1.6 (m, 4H,CH₂—CH₂), 1.05, 1.03 (2×s, 2×3H, 2×CH₃).

Example 12 Synthesis of 4-keto-9-cis-retinoic Acid

To a solution of 4-hydroxy-9-cis-retinoic acid (16 mg, 0.05 mmole) inCH₂Cl₂ (1.5 ml) was added Dess-Martin reagent [see Dess and Martin in J.Org. Chem. 48:4155 (1983)] (42 mg, 0.1 mmole) in one portion at 25° C.After stirring for 5 minutes, the mixture was diluted with 10 ml ofether and to this was added saturated aqueous NaHCO₃ (5 ml) containingNa₂SO₃ (55 mg). The mixture was stirred for 20 minutes to dissolve thesolid and the layers separated. The ether layer was washed with H₂O (2×5ml), brine (5 ml) and dried (MgSO₄). The solvent was recovered underreduced pressure and residue was subjected to flash columnchromatography (silica, 60% ether in Hexane) to give4-keto-9-cis-retinoic acid (14 mg. 90%), characterized as follows: TLCrf=0.6 (silica, 80% ether in hexane); ¹HNMR (400 mHz, CDCl₃) δ 7.05 (dd,J=16, 12 Hz, 1H, olefinic), 6.82 (d, J=16 Hz, 1 H, olefinic), 6.32 (d,J=16 Hz, 1H, olefinic), 6.30 (d, J=16 Hz, 1H, olefinic), 6.20 (d, J=12Hz, 1H, olefinic), 5.80 (s, 1H, olefinic), 2.5 (t, J=7 Hz, 2H, CH₂—CO),2.31 (s, 3H, CH₃), 2.01 (s, 3H, CH₃), 1.9 (s, 3H, CH₃), 1.89 (m, 2H,CH₂), 1.20 (s, 6H, 2×CH₃)

Example 13 In vitro Evaluation of 9-phenyl-9cis-retinoic Acid,4-hydroxy-9-cis-retinoic Acid and 4-keto-9-cis-retinoic Acid

The potency and efficacy of the compounds described in Examples 10, 11and 12 were determined (as described in Example 1—under the heading“Cotransfection Assay in CV-1 Cells”. The results are presented in Table4:

TABLE 4 9-cis-retinoic 9-phenyl-9-cis- 4-hydroxy-9-cis- 4-keto-9-cis-acid retinoic acid retinoic acid retinoic acid Potency Potency PotencyPotency Receptor (nM) Efficacy (nM) Efficacy (nM) Efficacy (nM) EfficacyRXRα 88 170% 210   76% 1700 161% 520 104% RXPβ 61 106% 44   88% 650 143%1300 105% RXRγ 360 137% 290   77% 1700 115% 1100 133% RARα 99 94% >10,000 <2% 380  65% 200  50% RAPβ 22  97% 880   39% 160  71% 26 67% RARγ 43 108% 250   59% 180  81% 55 107%

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

1. A composition in unit dosage form for oral administration, saidcomposition consisting essentially of: as an active ingredient, acompound selected from the group consisting of 9-cis retinoic acid,pharmaceutically acceptable salts thereof, and pharmaceuticallyacceptable hydrolysable esters thereof, and a pharmaceuticallyacceptable carrier suitable for oral administration.
 2. A composition inunit dosage form for oral administration, said composition consistingof: as an active ingredient, a compound selected from the groupconsisting of 9-cis retinoic acid, pharmaceutically acceptable saltsthereof, and pharmaceutically acceptable hydrolysable esters thereof,and a pharmaceutically acceptable carrier suitable for oraladministration.